This invention relates to an electrophoretic separation apparatus and in particular, to apparatus for treatment or separation of compounds including macromolecules in small volumes.
In the past, the separation of macromolecular solutes was performed by a process known as electrophoretic separation or electrophoresis, in particular, fixed boundary electrophoretic separation. In fixed boundary electrophoresis, a semi-permeable membrane (hereinafter referred to as a separation membrane), acts to separate two streams of liquid carrying macromolecular solutes such as proteins, known as the sample and the downstream. The streams flow between charged electrodes and at least one macromolecular solute migrates across the membrane from one stream to the other stream under the influence of the electric field. The apparatus also includes flow paths for buffer solutions and further semi-permeable membranes, hereinafter referred to as restriction membranes, disposed either side of the separation membrane between the electrodes and the separation membrane to separate the buffer flow paths from the sample and downstream. The restriction membranes allow the passage of ions but not of the relatively larger macromolecules.
Attempts have been made to improve upon such fixed boundary electrophoresis technology. In particular, one such improvement provided a system in which the separation and restriction membranes and sample and separation are contained in a removable and replaceable cartridge. Although this technology provides a substantial improvement, electrophoresis cannot satisfactorily be used for separating very small samples. Even an apparatus of reduced size is unable to separate very small samples, with the smallest practicable sample size being around 6 mL. Therefore, for smaller samples, other separation methods such as chromatography or gel electrophoresis, have to be used. However, such other methods are time consuming. For example, in gel electrophoresis, the separation in the gel is very slow, taking several hours and further time is wasted in subsequently extracting (eluting) the separated molecules from the gel. A further problem arises in that molecules tend to elongate and denature when separated in a gel in comparison with a zonal electrophoresis separation.
Earlier electrophoretic separation apparatus and methods have been developed for processing large sample volumes and are not suitable to treat small volumes. Furthermore, the ratio of sample to electrophoresis membrane surface area is usually greater than 2.5 mL/cm2, typically around 5 mL/cm2, which results in large dead volumes and the need to re-circulate buffers and samples to reduce heating and prevent clogging of membranes and other separation media.
The present invention seeks to alleviate these problems and in particular, to provide a separation apparatus and methods suitable for use with relatively small sample volumes.